The present invention relates to methods and compositions for regulating gene expression in a cell. In particular, the invention provides adjustable-threshold switch constructs which can selectively express or repress the expression of a gene of interest upon application of a threshold amount of an activating agent.
Many areas of biotechnology involve regulating the expression of one or more genes of interest by applying an external agent. Typical approaches for regulating gene expression involve natural or engineered transcription factors that activate or inhibit expression of a specific gene in response to a chemical agent [Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89:5547, 1992; Rivera, et al., Nat. Med., 2:1028, 1996; Yao and Evans, Proc. Natl. Acad. Sci. USA, 93:3346, 1996; Wang, et al., Proc. Natl. Acad. Sci. USA, 91:8180, 1994]. Transcription factors often are introduced into a cell using DNA constructs that express a transcription factor and a gene of interest. Stable activation or inhibition of a transcription factor typically requires a continuous application of a chemical agent, and the level of activation or inhibition generally is proportional to the amount of agent applied, over a range of agent concentrations. However, a concentration dependant response to an activating agent often results in inconsistent expression levels in experimental, industrial, or clinical settings. The instability of many agents and their susceptibility to experimental conditions often results in variations in the amount of active agent that is added to an expression system. This can result, for example, in variations in the amount of gene activation in a system where reproducible results are desired. Therefore, there is a need in the art for methods and compositions that are useful to reproducibly obtain a chosen level gene expression.
The invention provides methods and compositions that are useful to switch gene expression between two different expression states. According to the invention, the expression of one or more genes is changed from a first expression state to a second expression state by providing a preselected amount of an agent equal to or greater than a specific concentration (threshold concentration). When the agent is removed, or its concentration drops below the threshold concentration, the switch returns back to its first expression state. An important feature of the invention is that the amount of an agent required to switch gene expression between states may be adjusted by altering the threshold concentration of the switch. The threshold concentration is determined by the properties of the genetic components used to construct the invention, as described herein. In preferred switches of the invention, one or more genes are minimally expressed, in the first expression state, while the same gene or genes are maximally expressed in the second expression state. Accordingly, an adjustable threshold switch of the invention acts to switch gene expression between xe2x80x9conxe2x80x9d and xe2x80x9coffxe2x80x9d states.
The invention provides a recombinant adjustable-threshold switch that is characterized by two alternative expression states. In a first state, a first, inducible, promoter is substantially inactive and a second, constitutive, promoter is active. In a second state, the inducible promoter is active and the constitutive promoter is substantially inactive. Therefore, in the first state, genes that are transcribed from the constitutive promoter are expressed, and in the second state, genes that are transcribed from the inducible promoter are expressed. According to the invention, the application of the agent causes a transition from the first expression state to the second expression state if it is provided at or above the threshold concentration. If the concentration of agent drops below this level, the adjustable threshold switch returns back to the first expression state. Preferably, the transition from the first expression state to the second expression state does not occur in the absence of the agent.
With reference to FIG. 1A, the components of the recombinant adjustable threshold switch of the invention include a first nucleic acid (DNA or RNA) construct comprising a first regulatory gene (R1) that is expressed from a first, inducible promoter (P1), and a second nucleic acid construct comprising a second regulatory gene (R2) that is expressed from a second, constitutive promoter (P2). A product of the first regulatory gene inhibits or reduces (represses) expression of the second regulatory gene, and a product of the second regulatory gene inhibits or reduces (represses) expression of the first regulatory gene. According to preferred embodiments, either regulatory gene can be maximally expressed, but both preferably are not maximally expressed simultaneously. In a default first expression state, the constitutive promoter (P2) is active, and the inducible promoter (P1) is inactive (due to the absence of a sufficient amount of an activating agent and the inhibitory effect of the second regulatory gene product). The addition of an agent activates the inducible promoter (P1) to switch the threshold switch of the invention to a second expression state in which the inducible promoter (P1) is active and the constitutive promoter (P2) is substantially inactive. However, an important feature of the invention is that an amount of the agent equal to or greater than the threshold concentration is required to cause the transition to the second expression state. In the presence of a concentration of agent (for example, activating agent) equal to or greater than the threshold concentration, expression from the inducible promoter (P1) is sufficient to inhibit expression from the constitutive promoter (P2). While the concentration of the agent is maintained above the threshold concentration, the second expression state is maintained. When the concentration of the activating agent falls below the threshold concentration, the first expression state is reestablished.
According to the invention, the inhibitory effects of the regulatory gene products expressed from the inducible and constitutive promoters is dependent on both the expression level of the gene product and the inherent inhibitory properties of the gene product. Expression level is a function of promoter strength, RNA stability, translational efficiency (if the gene product is a protein), and protein stability (if the gene product is a protein). Accordingly, an agent that increases the inhibitory effect of the regulatory gene product expressed by the inducible promoter can cause a switch to another expression state if the agent is provided at or above a threshold concentration. Preferably, an agent that increases the expression level of the inducible regulatory gene product by increasing transcription, translation, RNA stability, protein stability, or a combination of the above, can cause a switch in expression states if it is provided at or above a threshold concentration.
According to the invention, one or more genes of interest can be linked to either one or both of the inducible and constitutive promoters of the invention. Accordingly, expression of a gene of interest can be activated or inhibited by the presence of a threshold amount of the agent. In a preferred embodiment, a gene of interest is transcribed from either of the inducible or constitutive promoters of the threshold switch construct. Alternatively, the gene of interest can be transcribed from a separate promoter that is identical to one of the two promoters of the switch, or that is regulated in the same way as one of the promoters of the switch.
According to the invention, useful agents include natural or synthetic chemical or biological molecules, and physical conditions such as temperature, UV exposure, or osmotic pressure. Such an agent stimulates the expression or inhibitory properties of the regulatory gene product expressed from the inducible promoter as described above.
In preferred embodiments of the invention, minor variations in the amount of an agent do not cause variations in the amount of expression of a gene of interest that is regulated by an adjustable-threshold switch. The important determinant of the expression state of the gene of interest is whether the concentration of the agent is above or below the threshold concentration.